FoxP3+ Treg cells help maintain immunologic tolerance and control inflammation in many contexts, as shown by the devastating multi-organ inflammation of FoxP3-deficient mice or human IPEX patients, and the discovery of Treg cells of different Treg subphenotypes playing diverse anti-inflammatory roles. The transcription factor FoxP3 specifies many aspects of Treg differentiation and function, but several other transcriptional cofactors and epigenetic modifiers partake in specifying the Treg lineage and its different effector functions. To tackle how FoxP3 orchestrates these activities, we constructed a broad set of mutants that span the entirety of the FoxP3 protein. Assessment of their transcriptional consequences revealed a finely variegated patchwork of mutation effects, providing detailed leads on FoxP3's functional facets, identifying classes of mutations with related impact, and parsing FoxP3 targets into clusters that depend on the same interactors. We will build on these data to chart FoxP3's physical interactions through the impact of the mutations, measuring changes in specificity to DNA sequence motifs, and using multiplexed mass spectrometry to identify changes in FoxP3-associated proteins. FoxP3 mutants will also be co-transfected with known co-factors to see which mutations abolish synergistic effects. This framework will be used to analyze the transcriptional effect of missense FoxP3 mutations from IPEX patients, introducing these mutations into the same transfection system to compare their transcriptional and physical effects to the mutant classes already identified. In collaboration wit colleagues at Children's (Boston) and Necker (Paris), these results will be complemented by profiling, with high-throughput single-cell RNA sequencing, ex vivo CD4+FOXP3+ Treg-like cells from patients with the same mutations. Finally, we will analyze the impact of the FoxP3 mutant classes in vivo, harnessing the speed and efficiency of CRISPR-based germline mutagenesis in mice to test, in the setting of Treg cells in vivo, six to eight mutations chosen to represent the main mutants classes and IPEX mutations. Analyzing immunological homeostasis in the mutant mice will show how perturbing FoxP3's interactions affects the differentiation and function of Treg cells, and their ability to control tolerance and autoimmune disease. This will reveal, in a tractable and non-confounded system, the consequences of the mutations from IPEX patients. These results will provide a mechanistic understanding of the pathways through which Foxp3 partakes in the control of different flavors and functions of Treg cells, and provide a new understanding of mutations in IPEX patients.